This invention relates to the use of a recombinant protein as receptor for a hepatitis virus, a nucleic acid coding for it, a vector comprising said nucleic acid, a host cell transformed or transfected with said nucleic acid, a new recombinant protein possessing the biological property to act as receptor for a hepatitis virus, and a transgenic animal expressing said recombinant protein.
In particular, the present invention relates to the use of a recombinant protein as receptor for a hepatitis B virus, a nucleic acid coding for it, a vector comprising said nucleic acid, an host cell transformed or transfected with said nucleic acid, a new recombinant protein possessing the biological property to act as receptor for a hepatitis B virus, and a transgenic animal expressing said recombinant protein.
It is known that the hepatitis B disease is caused by the HBV virus (Hepatitis B Virus), belonging to the family named hepadnaviruses, that are viruses possessing a DNA molecule as genome capable to infect differentiated hepatocytes. Such viruses is able to infect humans only and higher primates in the evolutive scale (Ganem D. et al., xe2x80x9cAnn. Rev. Biochem.xe2x80x9d, 56, 651-693, 1987).
The mechanism by which this non-cytolytic virus infects cells and induces liver damage, has not been completely understood up to now.
It is also known that the HBV has a genome endowed with unusual features of a small DNA molecule that is only partly double-strand and shows a single-strand region. The nucleocapsid, that encompasses the DNA, is essentially constituted by the core protein, also named HBcAg, which is able to interact with the viral DNA, and by a protein kinase able to phosphorylate the HBcAg protein. The virus has an envelope outside the core essentially composed of lipids and of three proteins, named Small (S), Medium (M) and Large (L), that constitute the surface antigen (HBsAg) of said virus (Tiollais P. et al., xe2x80x9cNaturexe2x80x9d, 317, 489-495, 1985).
The viral envelope plays an essential role in the interaction process of the virus with the receptor protein occurring on the hepatocyte plasma membranes, since the virus/receptor interaction represents the first step of infectious process. In particular, it has been shown that the N terminal region, also named preS1, of the L viral protein is responsible for the virus/receptor interaction and indispensable for the entry of said virus into the hepatocyte (Neurath A. R. et al., xe2x80x9cCellxe2x80x9d, 46, 429-436, 1986).
At least 350 million persons are chronic carriers of hepatitis B. The chronic disease occurs, in part, in patients with acute hepatitis B infections and, almost always, in cases of asymptomatic infection. This latter situation occurs in particular in the children infected from the mothers at the birth, where the disease symptoms became evident only when the chronic state is already reached. In about 25-35% of chronic carriers, the disease develops irreversible hepatic complications, due to chronic active hepatitis, cirrhosis or primary hepatocarcinoma, that lead to the death of more than 1 million persons per year.
Up to now, a specific pharmacological therapy to treat hepatitis B patients did not exist (Gitlin N, xe2x80x9cClin. Chem.xe2x80x9d, 43, 1500-1506, 1997).
In the case of chronic carriers, for which it is important to intervene to avoid permanent hepatic damage, the only treatment, used up to now, is the administration of xcex1-interferon. xcex1-interferon shows a wide range of properties such as antiviral, antiproliferative, immunomodulatory, cytostatic and/or cytotoxic.
However, xcex1-interferon therapy gives appreciable results only in the 30-40% of treated patients with chronic hepatitis. Furthermore, it is not possible to administer said molecule to all chronic carriers such as, for example, to patients showing high hepatic decompensation or portal hypertension or who are affected by autoimmune diseases.
The high species-specificity of HBV has not allowed developing an experimental model to study the infective process. In fact, the experimental system more utilized to study the HBV/hepatocyte interaction is represented by the use of human hepatocarcinoma cell line and named HepG2 (Knowles B. B. et al., xe2x80x9cNaturexe2x80x9d, 209, 497-499, 1980).
Cells of HepG2 line have the advantage of not displaying the genome of integrated HBV, the ability to bind an HBV virus and to allow the entry of viral DNA into cytoplasm, thus showing the presence, on the plasmatic membrane, of a receptor molecule able to bind and internalize said virus. (Neurath A. R. et al., xe2x80x9cCellxe2x80x9d, 46, 429-436, 1986; Dash S. et al., xe2x80x9cJ. Med. Virol.xe2x80x9d, 37, 116-121, 1992).
However, the use of this cell line as the experimental model has the disadvantage that the virus is not able to replicate itself and thus generate new viral particles.
Given the high need to have a molecule with the biological properties to act as receptor for a hepatitis B virus, up to now, different molecules able to interact with the outer envelope of HVB have been tested, such as, for example, the 6-Interleukin (Neurath A. R. et al, xe2x80x9cJ. Exp. Med.xe2x80x9d 172, 461-469, 1992), the ASGPR receptor (Asialoglycoprotein) (Trechel U. et al., xe2x80x9cJ. Gen. Virol.xe2x80x9d, 75, 3021-3029, 1994), the receptor for the IgA (Fcxcex1R) (Pontisso P. et al.,xe2x80x9cJ. Gen. Virol.xe2x80x9d 73, 2041-2045, 1992), the GAPD (Glyceraldehyde-3-P-Dehydrogenase) (Petit M. A. et al., xe2x80x9cVirologyxe2x80x9d, 187, 211-222, 1992), the pHSA (polymerized Human Sera Albumin) (Pontisso P. et al., xe2x80x9cJ. Virol.xe2x80x9d, 63, 1981-1988, 1989), the HSSP (Human Soluble Serum Protein) (Bubkowska A. et al., xe2x80x9cJ. Virol.xe2x80x9d, 67, 4316-4322, 1993), the endonexin II (Hertogos K. et al., xe2x80x9cVirologyxe2x80x9d, 197, 549-557, 1993), the ApoH (Apolipoprotein H) (Mehdi H. et al., xe2x80x9cJ. Virol.xe2x80x9d, 68, 2415-2424, 1994).
However none of the above mentioned proteins has shown definitive and specific receptorial activity for the viral particles. Indeed a biological assay able to evidence the receptorial function of a protein is not available. Such biological assay should comprise the transfection of a cDNA coding for a protein, in a suitable cell line, and the subsequent verification of the susceptibility to the infection for cells expressing the recombinant receptor (Mendelsohn L. C. et al., xe2x80x9cCellxe2x80x9d, 56, 855-865, 1989; Bergelson J. M. et al., xe2x80x9cSciencexe2x80x9d, 275, 1320-1323, 1997).
Now, it has been unexpectably found that the human protein SCCA1 (Squamous Cell Carcinoma Antigen) of FIG. 1 (SEQ ID NOS: 1 and 3) has the ability to act as HBV receptor.
In addition, the inventors have also found an new allelic variant of SCCA1 (FIG. 2; SEQ ID NOS: 2 and 4).
Therefore, it is a first object of this invention to provide the use of a recombinant protein having, fully or in part, a primary structure as shown in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof, as a receptor for a hepatitis virus.
Preferably, said virus is a hepatitis B virus.
Recently, the methodologies of molecular screening based on combinatorial library have been used to identify new molecules with different biological activity, as new drugs. Typical example of such methodology is reported in Kenan D. L. et al (xe2x80x9cTibsxe2x80x9d, 2, 57-64, 1994). For the system HBV virus/receptor, such methodologies allow to the person skilled in the field to select, from a very high number of molecules, those able to neutralize the virus/receptor interaction and to evaluate subsequently their applicability in the pharmacological field.
It is therefore another object of the present invention to provide the use of a recombinant protein having, fully or in part, a primary structure as shown in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof, or of any its derivative having the biological property to act as a receptor for a hepatitis virus, in molecular screening assays.
The inventors"" identification of a HBV receptor opens, the way to develop molecules with pharmacological activity able to neutralize the virus/receptor interaction occurring on the plasma membranes of hepatocytes and thus to inhibit the disease progression. This is obtained with methodologies known to the person skilled in the field. These are, for example, the use of the protein receptor as drug or as reagent for antibody production (monoclonal or polyclonal).
It is therefore a further object of present invention to provide the use of a recombinant protein having, fully or in part, a primary structure as shown in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof, or of any its derivative having the biological property to act as receptor for a hepatitis virus, as a drug.
Preferably, said drug is useful in the treatment of hepatitis B.
For monoclonal antibody production, for example, the procedure of Harlow and Lane may be ussed [xe2x80x9cAntibodiesxe2x80x9d, xe2x80x9cA Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)] and the different monoclonal antibodies may be tested in clinical trials to evaluate their activity in different pathologies such as, for example, cancer (Bodey B. et al., xe2x80x9cAnticancer Res.xe2x80x9d, 16, 661-674, 1996,), autoimmune diseases (Burmester G. R., xe2x80x9cBaillieres Clin. Rheumatol.xe2x80x9d, 6, 415-434, 1992), and viral diseases (Co M. S. et al, xe2x80x9cProc. Natl. Acad. Sci.xe2x80x9d, 88, 2869-2873, 1991).
It is, therefore, another object of the present invention to provide the use of a recombinant protein having, fully or in part, a primary structure as shown in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof, or of any its derivative having the biological property to act as receptor for a hepatitis virus, in the production of monoclonal antibodies.
It is also an other object of the present invention to provide the use of a recombinant protein having, fully or in part, a primary structure as shown in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof, or of any its derivative having the biological property to act as receptor for a hepatitis virus, in the production of polyclonal antibodies.
In addition, the complete sequence of a nucleic acid (FIG. 2; SEQ ID NO: 2) able to code for a protein having the biological property to act as receptor for a hepatitis B virus has been synthesized starting from the nucleic acid sequence of the SCCA1.
It is, therefore, an other object of the present invention to provide an isolated nucleic acid that comprises a nucleic acid coding for a protein having, fully or in part, a primary structure showed in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof, provided, however, that said nucleic acid is not the nucleic acid showed in FIG. 1 (SEQ ID NO: 1).
Preferably, said nucleic acid codes for a protein having the biological property to act as receptor for a hepatitis B virus.
For the production of a recombinant protein having the biological property to act as receptor for a hepatitis B virus, expression systems both in prokaryotic and eukaryotic hosts may be used.
For the expression in prokaryotes, the nucleic acid coding for the recombinant protein is cloned in a suitable expression vector, such as for example pET21, and then introduced in a suitable bacterium, such as, for example E. Coli. 
For the expression in eukaryotes, the nucleic acid coding for the recombinant protein, cloned in a suitable expression vector that allows stable or transient high expression levels, such as for example pcDNA3, is introduced in one of the large number of suitable mammalian cells.
It is therefore a further object of the present invention to provide a vector that comprises a nucleic acid coding for a protein having, fully or in part, a primary structure as shown in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof, provided, however, that said nucleic acid is not the nucleic acid shown in FIG. 1 (SEQ ID NO: 1).
It is also an object of the present invention to provide a prokaryotic or eukaryotic host cell permanently transformed or transfected with the above mentioned vector.
It is then an object of the present invention to provide an eukaryotic host cell constituting of the HepG2 cell line permanently transfected or transformed with a nucleic acid coding for a protein having, fully or in part, a primary structure as shown in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof.
It is a further object of the present invention to provide a recombinant protein having, fully or in part, a primary structure showed in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof, as a receptor for a hepatitis virus, provided, however, that the primary structure of said protein is not the protein as shown in FIG. 1 (SEQ ID NOS: 1 and 3).
The person skilled in the field can easily and routinely use the nucleic acid coding for the receptor protein for the hepatitis virus to generate transgenic animals, such as for example mice, expressing the receptor protein on the hepatocytes surface and thus inducing, in the animal, the human hepatitis infection.
Such animal models assume a remarkable pharmacological interest since they offer the possibility of an in vivo evaluation of the effect of the novel compounds with antiviral activity. Typical example of a known methodology for the generation of transgenic animals is described in Hogan et al. (xe2x80x9cManipulating the mouse embryoxe2x80x9d, Cold Spring Harbor Laboratory, 1986).
It is therefore another object of the present invention to provide a transgenic animal that is not an human being, characterized in that it expresses a recombinant protein having, fully or in part, a primary structure showed in FIG. 1 (SEQ ID NOS: 1 and 3) or 2 (SEQ ID NOS: 2 and 4), or of any allelic variant thereof.
Preferably, the above mentioned recombinant protein has the biological property to act as receptor for a hepatitis virus, and more preferably said receptor is a receptor for a hepatitis B virus.